The chemokine receptor, CXCR4, is involved in cancer growth, invasion, and metastasis. Several promising CXCR4 antagonists have been shown to halt tumor metastasis in preclinical studies, and clinical trials evaluating the effectiveness of these agents in patients with cancer are ongoing. However, the impact of targeting CXCR4 specifically on immune cells is not clear. Here, we demonstrate that genetic deletion of CXCR4 in myeloid cells (CXCR4^MyeΔ/Δ) enhances the antitumor immune response, resulting in significantly reduced melanoma tumor growth. Moreover, CXCR4^MyeΔ/Δ mice exhibited slowed tumor progression compared with CXCR4^WT mice in an inducible melanocyte Braf^V600E/Pten^−/− mouse model. The percentage of Fas ligand (FasL)–expressing myeloid cells was reduced in CXCR4^MyeΔ/Δ mice as compared with myeloid cells from CXCR4^WT mice. In contrast, there was an increased percentage of natural killer (NK) cells expressing FasL in tumors growing in CXCR4^MyeΔ/Δ mice. NK cells from CXCR4^MyeΔ/Δ mice also exhibited increased tumor cell killing capacity in vivo, based on clearance of NK-sensitive Yac-1 cells. NK cell–mediated killing of Yac-1 cells occurred in a FasL-dependent manner, which was partially dependent upon the presence of CXCR4^MyeΔ/Δ neutrophils. Furthermore, enhanced NK cell activity in CXCR4^MyeΔ/Δ mice was also associated with increased production of IL18 by specific leukocyte subpopulations. These data suggest that CXCR4-mediated signals from myeloid cells suppress NK cell–mediated tumor surveillance and thereby enhance tumor growth. Systemic delivery of a peptide antagonist of CXCR4 to tumor-bearing CXCR4^WT mice resulted in enhanced NK-cell activation and reduced tumor growth, supporting potential clinical implications for CXCR4 antagonism in some cancers. Cancer Immunol Res; 6(10); 1186–98. ©2018 AACR.